1. Field of the Invention
This invention relates to symplectin, which is a photoprotein derived from okinawan squid (Symplectoteuthis oualaniensis: Tobi-Ika). Moreover, this invention relates to an amino acid sequence of symplectin and a base sequence of gene encoding the protein. Furthermore, the present invention relates to a method for detection of a monovalent cation by monitoring luminescence, generated using said photoprotein in the presence of a chromophore.
2. Prior Art
Bioluminescence has been applied for various purpose, such as monitoring of concentration of a metal ion in a living cell, because some complementary factor is requisite for generation of luminescence. A chromophore is oxidized and then a high energy state intermediate (luminescent intermediate) is formed. It collapses to form a basal state and emission of luminescence occurs accompanied with such alteration of energy state. In a photogenic organism, emission of luminescent occurs efficiently using an enzyme.
The most classical bioluminescence is observed in firefly, which is well-known as luciferin-luciferase reaction. In luciferin-luciferase reaction, luciferin is converted to oxyluciferin via enzymatic reaction of luciferase in the presence of ATP and magnesium ion. It is the essential phenomenon involved in the photo-reaction. As the reaction mediated by firefly luciferase enables detection of luminescence with extremely high sensitivity, it is an important tool for investigation in the field of biochemistry. The gene encoding firefly luciferase has been already cloned. Then, heat-stable type luciferase is produced by genetic engineering technique using E. coli and it is commercially available.
For another example, aequorin, a blue fluorescent protein of jellyfish (Aequorea victoria) capable of emitting blue color luminescence, has been also known. Aequorin has a relatively low molecular mass of 21 kDa. Aequorin uptakes a chromophore such as coelenterazine and oxygen, then it is converted to an exited state (high energy state) using calcium ion as a trigger, thereby emits blue color luminescence. The color of jellyfish luminescence is actually blue color and not green color. However, green fluorescent protein (GFP) is assumed to receive energy from aequorin, thereby emitting green color luminescence.
The above luminescent system of jellyfish has been applied in the field of cell physiology and biochemistry. Emission of luminescence caused by aequorin is triggered by calcium ion. Then aequorin has been utilized to detect alteration of calcium ion concentration, such as for analysis on alteration of cytoplasmic calcium ion concentration caused by agonistic stimulation of intact cells, for example, by various hormones, agonists such as neurotransmitters or growth factors. Therefore, aequorin has attracted attention as a calcium sensor or a gene reporter, in the field of clinical biochemistry and cell physiology.
The above-mentioned method, utilizing luciferin or aequorin, is an excellent method for its high sensitivity. However, the ions which serves as the trigger of luminescence are magnesium ion and calcium ion for luciferin and aequorin, respectively. These are both divalent cations. Thus, there has been no means which enables detection of a monovalent cation using a photoprotein. The method utilizing a photoprotein is advantageous, because its sensitivity is extremely high and it is free from danger like experiments using a radioisotope. Therefore, a photoprotein which can detect a monovalent cation has been desired.
Thus, the inventors have noticed bioluminescent system of okinawan squid (Symplectoteuthis oualaniensis: Tobi-ika) from Okinawa, Japan. The luminescence of the squid was trigged by monovalent cations such as potassium, sodium or etc. and the inventors investigated on the luminescent system. In the luminescent system of okinawan squid, a monovalent cation acts as a trigger. Thus, the luminescent protein derived from okinawan squid is considered to be an excellent tool for detection of a monovalent cation.
One aspect of this invention of a protein derived from Symplectoteuthis oualaniensis (okinawan squid) having the following characteristics:
(1) emitting luminescence at wavelength of 470 nm by binding to dehydrocoeleneterazine or a dehydrocoeleneterazine derivative in the presence of potassium ion or sodium ion:
(2) having a molecular mass of approximately 60 kDa analyzed by SDS-polyacrylamide gel electrophoresis:
(3) dissolving into a solvent containing KCl at a concentration of 0.6M or higher while maintaining ability to emit luminescence: and
(4) decomposing by trypsin digestion to produce fragments having molecular masses of 40 kDa and 15 kDa.
According to the present invention, said dehydrocoeleneterazin derivative can be a compound selected from the group consisting of compounds represented by the following formula (1), (2) or (3): 
wherein X is a halogen atom or methoxy group, a compound represented by the following formula: 
wherein X represents a halogen atom, methoxy group or hydroxyl group, and Y and Z each represents hydrogen atom, a halogen atom, methoxy group or hydroxyl group, with the proviso that a case where both of Y and Z are hydrogen atoms is excluded, and
a compound represented by the following formula: 
wherein X and A each represents a halogen atom, methoxy group or hydroxyl group, and Y, Z, B and C each represents hydrogen atom, a halogen atom, methoxy group or hydroxyl group, with the proviso that cases where Y and Z are substituents selected from an atom or groups described above with both of B and C being hydrogen atoms and the case where all of Y, Z, B and C are hydrogen atoms are excluded.
Further aspect of this invention is a protein derived from Symplectoteuthis oualaniensis (okinawan squid) consisting of an amino acid sequence of following (a) or (b):
(a) an amino acid sequence represented by amino acid numbers 1 to 501 shown in SEQ:ID NO:1 in a sequence list, or (b) an amino acid sequence in which a part of said amino acid sequence (a) is deleted or another amino acid sequence is added to said amino acid sequence (a) or a part of amino acid sequence (a) is substituted with another amino acid sequence, the amino acid sequence (b) having characteristics that binds with dehydrocoeleneterazine by covalent bonding in the presence of potassium ion or sodium ion thereby emitting luminescence. The functional 40 kDa fragment of this protein, represented by amino acid numbers 1 to 370 shown in SEQ:ID NO:2 in a sequence list, is also within the range of this invention.
Moreover, further aspect of this invention is a method for detection of a monovalent cation, the method comprising formation of a conjugate adduct comprising the protein described above and an chromophore, thereby emitting luminescence from said conjugate adduct in the presence of a monovalent cation.
According to the present invention, said chromophore can be a compound selected from the group consisting of compounds represented by the following formula (4), (5), (6) or (7): 
compound represented by the following formula: 
wherein X is a halogen atom or methoxy group, a compound represented by the following formula: 
wherein X represents a halogen atom, methoxy group or hydroxyl group, and Y and Z each represents hydrogen atom, a halogen atom, methoxy group or hydroxyl group, with the proviso that a case where both of Y and Z are hydrogen atoms is excluded, and
a compound represented by the following formula: 
wherein X and A each represents a halogen atom, methoxy group or hydroxyl group, and Y, Z, B and C each represents hydrogen atom, a halogen atom, methoxy group or hydroxyl group, with the proviso that cases where Y and Z are substituents selected from an atom or groups described above with both of B and C being hydrogen atoms and the case where all of Y, Z, B and C are hydrogen atoms are excluded.
Moreover, further aspect of this invention is a gene derived from Symplectoteuthis oualaniensis (okinawan squid) consisting of a base sequence of following (e), (f) or (g):
(e) a base sequence represented by base numbers 1 to 1646 shown in SEQ:ID NO:3 in a sequence list,
(f) a base sequence in which a part of said base sequence (e) is deleted or another base sequence is added to said base sequence (e) or a part of base sequence (e) is substituted with another base sequence, the base sequence (f) encoding a protein having characteristics that binds with dehydrocoeleneterazine by covalent bonding in the presence of potassium ion or sodium ion thereby emitting luminescence, or
(g) a base sequence that hybridizes with the base sequence (e) under stringent conditions.
Moreover, further aspect of this invention is a method for detection of a monovalent cation, the method comprising introduction of the gene described above into a cell, expression of protein encoded by said gene in the cell, formation of a conjugate adduct of the protein and a chromophore, thereby emitting luminescence from said conjugate adduct in the presence of a monovalent cation.
According to the present invention, said chromophore can be a compound selected from the group consisting of compounds represented by the following formula (8), (9), (10) or (11): 
a compound represented by the following formula: 
wherein X is a halogen atom or methoxy group, a compound represented by the following formula: 
wherein X represents a halogen atom, methoxy group or hydroxyl group, and Y and Z each represents hydrogen atom, a halogen atom, methoxy group or hydroxyl group, with the proviso that a case where both of Y and Z are hydrogen atoms is excluded, and
a compound represented by the following formula: 
wherein X and A each represents a halogen atom, methoxy group or hydroxyl group, and Y, Z, B and C each represents hydrogen atom, a halogen atom, methoxy group or hydroxyl group, with the proviso that cases where Y and Z are substituents selected from an atom or groups described above with both of B and C being hydrogen atoms and the case where all of Y, Z, B and C are hydrogen atoms are excluded.
Moreover, further aspect of this invention is a transformed Escherichia coli wherein the gene described above is introduced.
Moreover, further aspect of this invention is a method to produce a recombinant protein, the method comprising introduction of the gene described above succeeded by expression of protein encoded by said gene in Escherichia coli, said recombinant protein binds with dehydrocoeleneterazine by covalent bonding in the presence of potassium ion or sodium ion to form a conjugate adduct, thereby emitting luminescence from said conjugate adduct.